Possible origin of sulfated glycosaminoglycans in human dental calculus

Abstract— The sulfated glycosaminoglycans present in human dental calculus have been shown to be dermatan sulfate and chondroitin-4-sulfate. The composition suggests that the glycosaminoglycans present in calculus, particularly subgingival material, could originate as a result of associated periodontal disease since closely similar compounds have previously been identified in normal and inflamed human gingiva.     

Isolation and identification of acid glycosaminoglycans in oral calculus

Samples of supra- and subgingival calculus were pooled, decalcified in EDTA and the organic matrix solubilized by autoclaving and proteolytic digestion. Acid glycosaminoglycans precipitated with ethanol and cetyl pyridinium chloride were studied both by chemical and electrophoretic techniques. Chondroitin sulphate, hyaluronic acid and possibly heparan sulphate were shown to be present in the organic phase. It is suggested that chondroitin sulphate may have a role in the mineralization of dental calculus as postulated for other calcified tissues.     

Glycosaminoglycans of human cementum

The glycosaminoglycans in human cementum have been studied. Following proteolytic digestion of guanidine/EDTA and collagenase extracts of cementum, glycosaminoglycans were isolated and then separated by cellulose acetate membrane electrophoresis. After specific elimination by enzymatic and chemical treatments the glycosaminoglycans were identified as hyaluronic acid, chondroitin sulfate and dermatan sulfate. Neither heparan sulfate nor keratan sulfate were observed. Quantitation of the glycosaminoglycans in both extracts revealed chondroitin sulfate to represent the major species present. Hyaluronic acid was observed predominantly in the guanidine/EDTA extract while dermatan sulfate was a quantitative minor component of both extracts.     

The crystalline components of dental calculi: human vs. dog.

Dental calculus from the dog was found to consist principally of the calcite form of calcium carbonate mixed with small amounts of apatite; other calcium phosphates, consistently present in human calculus, were not present in dog calculus. Precipitable calcium salts from human saliva were mainly apatite; for the dog the principal precipitated salt was calcium carbonate (calcite form).     

Inhibitory effect of a novel bisphosphonate, TRK-530, on dental calculus formation in rats

BACKGROUND: A newly developed bisphosphonate, TRK-530 (disodium dihydrogen[4-(methylthio)phenylthio]methanebisphosphonate), has recently been reported to show anti-inflammatory and anti-bone-resorbing activity. Since bisphosphonates have been shown to inhibit the formation of calcium-phosphate crystals in vitro, TRK-530 may inhibit the formation of dental calculus. Therefore, the present study was performed to examine whether this compound has such an effect. METHODS: Three groups of Wistar rats fed a calculogenic diet (RC16) were treated with TRK-530 in drinking water at concentrations of 0 (control group), 0.75, and 1.5 mM. Another group received a daily subcutaneous injection of TRK-530 at a dose of 2.25 micromoles/rat, which was assumed to correspond to the maximum amount of this compound absorbed from the intestine when rats received 1.5 mM TRK-530 in drinking water. Rat dental calculus formation was evaluated. The crystalline nature of dental calculus was studied by x-ray diffraction analysis. Finally, the effects of TRK-530 on the precipitation of calcium-phosphate from solution were tested in vitro. RESULTS: TRK-530 in drinking water inhibited dental calculus formation dose-dependently. However, subcutaneous injection of TRK-530 did not have any significant effect, suggesting that the anticalculus effect of TRK-530 in drinking water was topical, not systemic. The calculus that formed in both the control and experimental groups was primarily hydroxyapatite, a main constituent of human dental calculus. TRK-530 inhibited the precipitation of calcium-phosphate from solution in vitro. CONCLUSIONS: TRK-530 inhibited the formation of dental calculus in a dose-dependent fashion via a local effect. Inhibition of the precipitation of calcium-phosphate from solution might be involved in the anticalculogenic mechanism of this drug.     

Detection,removal and prevention of calculus: Literature Review

Dental plaque is considered to be a major etiological factor in the development of periodontal disease. Accordingly, the elimination of supra- and sub-gingival plaque and calculus is the cornerstone of periodontal therapy. Dental calculus is mineralized plaque; because it is porous, it can absorb various toxic products that can damage the periodontal tissues. Hence, calculus should be accurately detected and thoroughly removed for adequate periodontal therapy. Many techniques have been used to identify and remove calculus deposits present on the root surface. The purpose of this review was to compile the various methods and their advantages for the detection and removal of calculus.     

Site specific mineral composition and microstructure of human supra-gingival dental calculus

Dental calculus has been implicated in the aetiology of several periodontal conditions. Its prevention and removal are therefore desirable clinical goals. While it is known that calculus is very variable in chemical composition, crystallinity and crystallite size little is known about site specific variability within a dentition and between individuals. With this in mind, a study was undertaken to investigate the comparative site specific nature and composition of human dental supra-gingival dental calculus obtained from 66 male patients visiting for their dental check-up using fluorescent X-ray spectroscopy, X-ray diffractometry and Fourier transform infrared spectroscopy. The supra-gingival dental calculus formed on the lingual surfaces of lower anterior teeth and the buccal surfaces of upper molar teeth were classified into four types based on calcium phosphate phases present. There was significant difference in composition of the crystal phase types between lower and upper teeth (p<0.01). There was no significant difference in crystal size between dental calculus on anterior or molar teeth of all samples. The degree of crystallinity of dental calculus formed on the upper molar teeth was higher than that formed on the lower anterior teeth (p<0.01). The CO(3)(2-) contents in dental calculus formed on the lower anterior teeth were higher than on upper molar teeth (p<0.05) which might explain the difference in crystallinity. Magnesium and Si contents and Ca:P ratio on the other hand showed no significant difference between lower and upper teeth. It was concluded that the crystal phases, crystallinity and CO(3)(2-) contents of human dental supra-gingival dental calculus is related to its location in the mouth.     

Reduction of calculus accumulation in domestic ferrets with two dentifrices containing pyrophosphate

Work performed by King et al. in the 1940's and 1950's, as well as recent studies by our group, have shown that the domestic ferret is a suitable model for the study of calculus formation, offering several advantages over the rodent and dog models in current use. We have demonstrated that mineral supplementation of a moist diet accelerates calculus accumulation, and that twice-daily application of a regular dentifrice slows the initial rate of calculus formation, but permits significant accumulation by the eighth week. The present study compared calculus accumulation in female ferrets receiving mineral-supplemented cat food and a twice-daily application of either Regular Crest toothpaste (Crest), Anti-tartar Crest (Crest-AT), or Anti-tartar Colgate Gold toothpaste (Colg-AT). Animals received an ultrasonic prophylaxis, then were fed once daily for eight weeks with moist canned cat food supplemented with sucrose and mineral salts, and were scored for area and extent of calculus accumulation at four and eight weeks after prophylaxis. The data show that the groups treated with the anti-calculus dentifrices produced significantly less calculus than the group treated with regular dentifrice; the Colg-AT group also exhibited lower scores than did the Crest-AT group, especially at four weeks. These results, similar to those seen in human studies, demonstrate that the ferret is a suitable model for the study of anti-calculus dentifrices.     

Isolation and characterization of the proteoglycans synthesized by adult human pulp fibroblasts in vitro

The proteoglycans synthesized by fibroblasts derived from healthy human adult dental pulps have been isolated and characterized on the basis of their glycosaminoglycan content, molecular size and charge. The proteoglycans were identified by their labelling with [³⁵S] sulphate and susceptibility to digestion by papain. The sulphated glycosaminoglycans associated with the proteoglycans were identified following specific enzymatic and chemical degradations as chondroitin sulphate, dermatan sulphate and heparan sulphate. Dermatan sulphate and chondroitin sulphate were identified as the major glycosaminoglycans secreted into the medium, whereas chrondroitin sulphate and heparan sulphate were the principal glycosaminoglycans associated with the cell layers. The proteoglycans could be fractionated on the basis of their charge and size into a number of heterogeneous pools. The principal proteoglycans isolated were small and contained either chondroitin sulphate or dermatan sulphate and most likely correspond to decorin and biglycan. Other molecules with features similar to versican and syndecan were also identified.     

Molecular size distribution of proteoglycans in human inflamed gingival tissue

Proteoglycans were extracted from human gingiva with 2 M CaCl2. The extracts were examined by gel filtration on Sephacryl S-400 in 2 M CaCl2 under dissociative conditions. The 280 nm absorbance profiles of clinically uninflamed, inflamed and severely-inflamed tissues showed that material was present with molecular weights of between 2 X 10(6) or greater, and 16,000. Proteoglycans were examined by cellulose-acetate electrophoresis with subsequent identification of the constituent glycosaminoglycans after protease digestion, and finally by chondroitinase AC digestion of the liberated glycosaminoglycans. The relative proportion of each glycosaminoglycan was calculated by scanning each cellulose-acetate sheet on an integrating densitometer. Heparan sulphate was found only in fraction I (mol. wt 2 X 10(6) or greater), together with hyaluronic acid and chondroitin-4-sulphate, these being present in all of the glycosaminoglycan-containing fractions (I-IV). Dermatan sulphate was absent from fraction I, but present in II-IV, apparently existing on the same protein core as chondroitin-4-sulphate. The relative proportions of these two glycosaminoglycans was related to molecular size, and with the degree of inflammation for a given molecular species.     

Fluorescence spectroscopy of dental calculus

BACKGROUND: Correct diagnosis of the presence and extent of subgingival calculus is important for periodontal treatment planning and reassessment after periodontal therapy. Traditional tactile methods often lack sensitivity. The present investigation shall contribute to understanding the fundamental fluorescence properties that may be useful for optical detection of both supra- and subgingival calculus. OBJECTIVES: The aim of this study was to investigate emission spectra from supra- and subgingival calculus under a wide range of excitation wavelengths. METHODS AND RESULTS: Extracted human molars with either supragingival or subgingival calculus deposits on the root surface were selected (n = 3 each). Emission spectra were recorded from the calculus of each tooth and corresponding areas of clean root surfaces using a fluorescence spectrophotometer at excitation wavelengths from 360 nm up to 580 nm in steps of 20 nm. The spectra were corrected for the wavelength dependent instrument sensitivity and normalized to peak intensity (the highest peak was set at 1.0). Emission spectra of calculus exhibited distinct fluorescence bands between 570 and 730 nm not present in clean root surfaces. This fluorescence emission was strongest for excitation wavelengths from 400 to 420 nm. No differences were observed between supra- and subgingival calculus. CONCLUSIONS: Human dental calculus can clearly be differentiated from clean root surfaces by emission spectrophotometry. The characteristic fluorescence emission of supra- and subgingival calculus may be due to a variety of porphyrin derivatives and may provide the basis for future diagnostic procedures.     

Qualitative and quantitative analyses of bovine gingival glycosaminoglaycans.

Bovine gingival glycosaminoglycans have been analysed qualitatively and quantitatively by two-dimensional electrophoresis on a cellulose acetate strip. The four spots observed were identified as chondroitin 4-sulphate, dermatan sulphate, hyaluronic acid and heparan sulphate. Neither chondroitin 6-sulphate nor heparin and keratan sulphate were observed.The major components of bovine gingival glycosaminoglycans were chondroitin 4-sulphate, 32–40 per cent; dermatan sulphate, 33–37 per cent; hyaluronic acid, 17–27 per cent. Heparan sulphate was present only in a limited amount. The total uronic acid content of bovine gingiva, however, decreased with age, especially during the first three years of life, possibly due to the marked decrease of both chondroitin 4-sulphate and dermatan sulphate. After 3 years of age, the decrease of these glycosaminoglycans slowed down considerably. Hyaluronic acid decreased rather slowly from the time of birth to 10 years of age, and heparan sulphate decreased initially but increased later.     

Bacteria and archaea paleomicrobiology of the dental calculus: a review

Dental calculus, a material observed in the majority of adults worldwide, emerged as a source for correlating paleomicrobiology with human health and diet. This mini review of 48 articles on the paleomicrobiology of dental calculus over 7550 years discloses a secular core microbiota comprising nine bacterial phyla – Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, TM7, Synergistetes, Chloroflexi, Fusobacteria, Spirochetes – and one archaeal phylum Euryarchaeota; and some accessory microbiota that appear and disappear according to time frame. The diet residues and oral microbes, including bacteria, archaea, viruses and fungi, consisting of harmless organisms and pathogens associated with local and systemic infections have been found trapped in ancient dental calculus by morphological approaches, immunolabeling techniques, isotope analyses, fluorescent in situ hybridization, DNA‐based approaches, and protein‐based approaches. These observations led to correlation of paleomicrobiology, particularly Streptococcus mutans and archaea, with past human health and diet.     

Glycosaminoglycans of temporomandibular articular discs

abstract — The glycosaminoglycans of the fibrous cartilage of temporomandibular articular discs from rat, rabbit, dog and monkey have been separated by means of cellulose acetate membrane electrophoresis on a micro scale. The total glycosaminoglycan amount present in these tissues was found to be 1.8, 1.8, 1.4 and 1.2 μg uronic acid, respectively, per mg tissue wet weight. Two major glycosaminoglycan fractions with the electrophoretic mobility of hyaluronate and dermatan sulfate were present, with the latter predominating.     

Identification of calprotectin, a calcium binding leukocyte protein, in human dental calculus matrix

Calprotectin is a calcium binding protein produced by leukocytes, macrophages and epithelial cells, and its levels in several tissues increase during infections and in many inflamed areas, suggesting that it may be an indicator of inflammatory activity. Osteopontin is a prominent phosphorylated glycoprotein in bone matrix, having calcium binding capacity. Recently, it has been reported that calprotectin and osteopontin are present in urinary stones (pathological mineralized masses in the body), and that these proteins may be involved in their formation. Dental calculus formed by mineralization of dental plaque is an inflammatory factor which may contribute to periodontal disease. It contains many organic components involved in mineralization. We recently found osteopontin molecules in human dental calculus and suggested that the components of its matrix may be similar to those of urinary stones. In this study, we investigated the presence of calprotectin in human dental calculus by immunohistochemical and immunoblotting analyses using a specific antibody for calprotectin. After fixation and demineralization of dental calculi adhered to tooth roots, sections embedded in paraffin were immunoreacted with the antibody for calprotectin and positive immunostaining for calprotectin was observed. Dental calculus proteins were then extracted with EDTA and separated by electrophoresis on 15% polyacrylamide gels. By immunoblotting analysis, 3 or 4 bands were observed at 11, 14.5, 22–25, 28 or 36.5 kDa and these patterns corresponded to those of calprotectin subunits. When non-immune rabbit serum was used instead of calprotectin-specific antibody as a negative control, no immunoreactivity was observed. These findings indicate that calprotectin is associated not only with antibacterial action but also with calcium binding capacity during dental calculus formation.     

Laboratory animal models in periodontology

Animal models are needed to objectively evaluate the pathogenesis of human periodontal diseases and its various treatment modalities. Selection of the appropriate animal model depends on the similarity of the periodontium and the nature of the disease to that of humans. The more commonly used animal models for studying the pathogenesis of periodontal disease, use of implants and guided tissue regeneration have been dogs and nonhuman primates. Periodontal disease in rodents has not been found to be as closely related to the human varieties. Rats and hamsters are best suited for caries and calculus research. Ferrets may be a promising new model for studying periodontal disease and calculus formation. Variables unique to each animal species are manifested by a wide range of clinical and histopathological features. Different species have distinct diets, habits, life spans, tissue structures, host defense mechanisms and genetic traits. This article describes the diversity seen in animal models used to study microbiological, immunological, and clinical features of periodontal disease and its prevention and treatment.     

Oral features and dental health in Hurler Syndrome following hematopoietic stem cell transplantation

International Journal of Paediatric Dentistry 2010; 20: 322–329 Background. Hurler Syndrome is associated with a deficiency of a specific lysosomal enzyme involved in the degradation of glycosaminoglycans. Hematopoietic stem cell transplantation (HSCT) in early infancy is undertaken to help prevent the accumulation of glycosaminoglycans and improve organ function. Aim. To investigate the oral features and dental health of patients with Hurler Syndrome who have undergone successful HSCT. Materials and methods. Twenty‐five patients (median age 8.6 years) post‐HSCT (mean age 9.4 months) underwent oral assessment (mean of 7.5 years post‐HSCT). Results. Dental development was delayed. Numerous occlusal anomalies were noted including: open‐bite, class III skeletal base, dental spacing, primary molar infra‐occlusion and ectopic tooth eruption. Dental anomalies included hypodontia, microdontia, enamel defects, thin tapering canine crowns, pointed molar cusps, bulbous molar crowns and molar taurodontism. Tooth roots were usually short/blunted/spindle‐like in permanent molars. The prevalence of dental caries was low in the permanent dentition (mean DMFT 0.7) but high in the primary dentition (mean dmft 2.4). Oral hygiene instruction with plaque and or calculus removal was indicated in 71% of those that were dentate. Conclusion. Patients with Hurler Syndrome post‐HSCT are likely to have delayed dental development, a malocclusion, and dental anomalies, particularly hypodontia and microdontia.     

Isolation, identification, and quantitation of glycosaminoglycans synthesized by human gingival fibroblasts in vitro

The glycosaminoglycans synthesized by diploid fibroblasts obtained from healthy human gingivae of three donors were isolated, identified, and quantified. Degradation with specific enzymes identified the glycosaminoglycans as hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate; hyaluronic acid predominating. The distribution of the sulfated glycosaminoglycans in the cell layer and the medium was not the same. The cells contained mainly heparan sulfate (48.3%) and the medium mainly dermatan sulfate (47%).     

Calculus deposits and bone loss on the teeth of Romano-British and eighteenth-century Londoners

The relation between dental calculus and periodontal disease is not clear but it is generally recognized that calculus is a significant pathogenetic factor. Skeletal material has previously been used to study some aspects of chronic adult periodontitis but few studies have quantified the extent of calculus in ancient populations and its relation to changes in alveolar bone height. This study records the presence and extent of calculus and its relation to alveolar bone loss in a Romano-British and eighteenth-century London population. There were significant differences in calculus deposition in the two populations but this appeared to have little effect on changes in alveolar bone contour. It is suggested that the amount of calculus may be related to diet but that changes in alveolar bone height seem to be independently controlled.     

Molecular size distribution analysis of human gingival glycosaminoglycans in cyclosporin- and nifedipine-induced overgrowths

Glycosaminoglycans are thought to accumulate in formative lesions like drug-induced gingival overgrowth. Recent evidences, however, suggest that the amounts of glycosaminoglycans are comparable in overgrown and healthy gingiva. Besides, alterations in the size distribution of glycosaminoglycan molecules isolated from phenytoin-induced overgrown samples have also been suggested. Therefore, we sought to determine possible differences in molecular size distribution of gingival glycosaminoglycans in other types of drug-induced overgrowths. Purified gingival glycosaminoglycans from healthy and cyclosporin- and nifedipine-induced overgrown gingival tissues were analyzed by agarose gel electrophoresis and their molecular-size distribution was evaluated by both gel filtration chromatography and polyacrylamide gel electrophoresis. Our results on the gingival glycosaminoglycan composition showed presence of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid in all types of gingival tissues examined. In addition, hyaluronic acid was predominantly of a large size eluting near to the void volume of a Superose-6 column, while the sulfated glycosaminoglycans were mainly composed of low molecular size glycosaminoglycans. Our results show no differences in the molecular-size distribution of hyaluronic acid and sulfated glycosaminoglycans among healthy and drug-induced overgrown gingival tissues.